Methods of propagating monkey adenoviral vectors

ABSTRACT

The invention provides methods for propagating a monkey adenovirus in a cell including a human cell, comprising one or more gene products isolated from a human adenovirus. Also provided are methods for propagating wherein the monkey adenovirus comprises a nucleic acid sequence encoding a human adenovirus gene product. The invention further provides a monkey adenovirus. including a replication-deficient monkey adenovirus, obtained by such propagation methods.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is the U.S. National Stage under 35 U.S.C. §371 ofInternational Application No.: PCT/US2010/055991, filed Nov. 9 2010,which claims the benefit of U.S. Provisional Appl. No. 61/259,343, filedNov. 9, 2009, the content of which is incorporated by reference hereinin its entirety.

INCORPORATION-BY-REFERENCE OF MATERIAL SUBMITTED ELECTRONICALLY

Incorporated by reference in its entirety herein is a computer-readablenucleotide sequence listing submitted concurrently herewith andidentified as follows: One 38,463 Byte ASCII (Text) file named“SEQ_Listing.TXT,” created on Nov. 8, 2010.

FIELD OF THE INVENTION

The present invention relates to monkey adenoviruses, and methods forgrowing monkey adenoviruses, which enable their use in a variety ofapplications, including the treatment and prevention of human diseases.

BACKGROUND OF THE INVENTION

Recombinant eukaryotic viral vectors have become a preferred means ofgene transfer for many researchers and clinicians. In vivo gene transferis a strategy in which a nucleic acid, usually in the form of DNA, isadministered to effect expression of the protein product of thetransferred gene in a location that is beneficial to the recipient. Thebenefit can be the induction of an immune response to the gene product,i.e., vaccination, or modification of the genetic repertoire of targetcells for therapeutic purposes. This can be accomplished efficientlyusing a recombinant adenoviral vector encoding a so-called “transgene.”Adenoviral vectors have advantages over other vectors commonly employedfor gene transfer (e.g., retroviral vectors) since adenoviral vectors(i) can be produced in high titers (i.e., up to 10¹³ viralparticles/ml); (ii) they efficiently transfer genes to nonreplicating aswell as replicating cells; (iii) recombination is rare; (iv) there areno known associations of human malignancies with adenoviral infectionsdespite common human infection with adenoviruses; (v) the adenoviralgenome can be manipulated to accommodate foreign genes that range insize; (vi) an adenoviral vector does not insert its DNA into thechromosome of a cell, so its effect is impermanent and unlikely tointerfere with the cell's normal function; and (vii) live adenovirus,having as an essential characteristic the ability to replicate, has beensafely used as a human vaccine (Straus, In Adenoviruses, Pienan Press,New York, N.Y., 451-496 (1984); Horwitz et al., In Virology, 2nd Ed.,Fields et al., eds., Raven Press, New York, N.Y., 1679-1721 (1990);Berkner, BioTechniques, 6: 616 (1988); Chanock et al., IAMA, 195: 151(1966); HajAhmad et al., J. Virol., 57: 267 (1986); and Ballay et al.,EMBO J, 4: 3861 (1985)). The human adenovirus is one of the most widelyused recombinant viral vectors in current viral vectored vaccine andgene therapy protocols.

In terms of general structure, all adenoviruses examined to date arenonenveloped, regular icosahedrons of about 65 to 80 nanometers indiameter. Adenoviruses are comprised of linear, double-stranded DNA thatis complexed with core proteins and surrounded by the adenoviral capsid.The proteins of the capsid are the targets of neutralizing antibodiesand the different serotypes possess distinct amino acid sequences in thecapsid proteins that are on the outside of the viral particle.

Adenoviruses belong to the family Adenoviridae, which is divided intofive genera, Mastadenovirus, Atadenovirus, Siadenovirus, Aviadenovirus,and Ichtadenovirus. The adenoviruses in the genus Mastadenovirus infectmammals only and include the human, chimpanzee, and monkey adenoviruses.

Adenoviruses provide an elegant and efficient means of transferringtransgenes into cells. However, one problem encountered with the use ofadenoviral vectors for gene transfer in vivo is the presence ofpre-existing immunity to adenovirus that was acquired by the recipientthrough natural exposure to the adenoviruses. Primarily, infection withadenovirus throughout life induces the generation of antibodies toantigenic epitopes on adenoviral capsid proteins. If sufficient intiter, the antibodies can limit the efficacy of the adenovirus genetransfer vector. In addition, the administration of an adenovirus vectorcan induce immunity; thus an adenovirus may not be used more than onceas an effective gene transfer vehicle. For instance, animal studiesdemonstrate that intravenous or local administration (e.g., to the lung,heart or peritoneum) of an adenoviral type 2 or 5 gene transfer vectorcan result in the production of antibodies directed against the vectorwhich prevent expression from the same serotype vector administered 1 to2 weeks later (see, e.g., Yei et al., Gene Therapy, 1: 192-200 (1994);Zabner et al., Nat. Gen., 6: 75-83 (1994); Setoguchi et al., Am. J.Respir. Cell. Mol. Biol., 10: 369-377 (1994); Kass-Eisler et al., GeneTherapy, 1: 395-402 (1994); Kass-Eisler et a)., Gene Therapy, 3: 154-162(1996)). This is a drawback in adenoviral-mediated gene transfer, sincemany uses of an adenoviral vector (e.g., for inducing or boosting theimmune response to a pathogen or providing a second dose of atherapeutic) require repeat administration. The mechanism by whichantibodies directed against an adenovirus are able to prevent or reduceexpression of an adenoviral-encoded gene is unclear. However, thephenomenon is loosely referred to as “neutralization”, and theresponsible antibodies are termed “neutralizing antibodies.” Thus, totake full advantage of adenovirus vectors for in vivo gene transfer,novel types of adenoviruses are needed that (1) are not susceptible toneutralization by antibodies directed against another type, and (2) arenot susceptible to neutralization by antibodies commonly found in thehuman population.

There are many different adenoviruses isolated from a broad range ofanimal hosts and adenoviruses are named by host first isolated from.I-lost animals from which adenoviruses have been isolated includemammals, birds, snakes, frogs, and fish. The mammalian hosts include,among others, primates such as monkeys, humans, and chimpanzees.

Humans and chimpanzees are very closely related and are grouped togetheras hominids. In contrast, monkeys are not grouped with humans andchimpanzees because there is a significantly greater evolutionarydistance between them. The monkeys diverged between 25 and 35 millionyears ago from the hominids, whereas humans and chimpanzees divergedonly about 7 million years ago (Samonte and Eichler, Nature ReviewsGenetics, 3: 65-72 (2002)). These similarities and differences betweenhumans, chimpanzees, and monkeys are consistent with documented hostrange restrictions of adenoviruses.

Many different ways for host range restriction occur. For example,wild-type human adenoviruses do not grow productively on monkey cells.In monkey cells infected with wild-type human adenovirus, the viralearly genes are properly expressed (Feldman et al., J Bacteriol, 91:813-8 (1966); Van der Vliet and Levine, Nature, 246: 170-4 (1973)), andviral DNA replication occurs normally (Rapp et al., J Bacteriol, 92:931-6 (1966); Reich et al., PNAS, 55: 336-41 (1966); Friedman et al., J.Virol, 5: 586-97 (1970)). However, the expression of several late viralproteins is reduced. The block to late gene expression appears to be dueto abnormal production of the viral late mRNAs (Klessig and Anderson, J.Virol, 16: 1650-68 (1975)), and this block can be overcome by a singlemutation of the adenovirus DNA Binding Protein (DBP) (Klessig andGrodzicker, Cell, 17: 957-66 (1979)). Human adenoviruses that containthis single mutation in the DBP grow productively on monkey cells,suggesting that the key to the monkey/human block is centered on theroles of the DBP during the life cycle of the adenovirus.

In contrast to the monkey/human block are the observations thatadenoviruses isolated from chimpanzees do not have a restriction inhuman cells and can be propagated efficiently (W. P. Rowe et al., Proc.Soc. Exp. Biol. Med., 97(2): 465-470 (1958); W. D. Hillis et al.,American Journal of Epidemiology, 90(4): 344-353 (1969); N. Rogers etal., Nature, 216: 446-449 (1967)). In particular, replication of somechimpanzee adenovirus isolates was found to be more efficient in humanthan in monkey cells (M. Basnight et al., American Journal ofEpidemiology, 94(2):166-171 (1971)). Adenoviruses isolated from othergreat apes species, such as gorillas and bonobos, have also recentlybeen shown to grow in human cells (S. Roy et al., PLoS Pathogens, 5(7):e1000503 (2009)). Wild-type chimpanzee adenovirus replication in humancells does not require the expression of human adenovirus complementingfactors, since E1-expressing cell lines (e.g., human embryonic kidney293 cells, human retina PER.C6 cells) and non-expressing cell lines(A549 human lung epithelial carcinoma cells) have been used for theirpropagation (U.S. Pat. No. 6,083,716; S. F. Farina et al., Journal ofVirology, 74(23):11603-11613 (2001); S. Roy et al, Virology, 324:361-372 (2004); S. Roy et al., Human Gene Therapy, 15: 519-530 (2004);E. Fattori et al, Gene Therapy, 13(14):1088-1096 (2006); J. Skog et al.,Molecular Therapy, 15(12): 2140-2145 (2007); D. Peruzzi et al., Vaccine,27(9): 1293-1300 (2009)). The absence of a replication block isconsistent with the close evolutionary distance between the human andchimpanzee lineages, which diverged only about 7 million years ago(Samonte & Eichler, Nature Reviews Genetics, 3: 65-72 (2002)).

Consistent with the greater divergence of hosts, a host rangerestriction of monkey adenoviruses for growth on human cells has beendescribed (Am. J. Hyg., 68: 31 (1958); Virology, 35: 248 (1968);Savitskaya et al., Doklady Biochemistry, 375: 242 (2000); Alstein etal., JVi, 2: 488 (1968); Genetika, 39(6): 725-31 (June 2003)), and ithas been hypothesized that the determinants are partially E4 andpossibly E2. Savitskaya et al., supra, reported there was no growth ofthe monkey adenovirus SV7(C8) (now known as SV16 (ICTV 8^(th) Report, p.220)) on human embryonic kidney (HEK) cell line 293. Thus, an E1 regionfrom a human adenovirus was not sufficient to alleviate the block toreplication. The virus could grow on HEK-293 cells with Ad5 E4 regioninserted (VK-10-9 cells). However, the VK-10-9 cells provided onlypartial alleviation of the replication block since replication was40-fold lower than on CV1 cells (continuous line of green monkeykidney). This showed there was still a block to monkey virus replicationin VK-10-9 cells. The authors concluded that E4 expression was too low,based on E4 ORF3 protein level (Krougliak and Graham, Hum Gene Ther., 6:1575 (1995)), and a virus specific product was probably required(Savitskaya et al., supra). The authors then proposed the product mightbe encoded by the E2A gene, though additional study would be needed toclarify the problem. Savitskaya et al., supra, also notes that low E4expression could have been the cause or an additional factor from E2Awould be required for complete release of the replication block, andadditional study was needed to define the causes. Interestingly,although the level of E4 expression in the VK-10-9 cells was reported tohe significantly lower than that during wild type Ad5 replication, itwas high enough for replication of an E4-deleted human adenovirus type 5virus to the same level as wild type human Ad5 in HEK-293 cells(Krougliak and Graham 1995), further suggesting that the expressionlevel of E4 was not the complete explanation for the species-specificblock. Therefore, while it was believed that more E4 expression and/oran E2A product were required, neither was required. Also, it is apparentthat the Ad 5 E4 function required for virus growth is separate fromthat required to overcome host range restriction of monkey adenoviruseson human cells because the E4 requirement for growth is not the same forhost range determination. Thus, Savitskaya et al., supra, demonstratesthat adenovirus E1 and E4 regions are likely not sufficient foralleviating the species block, and that other regions, in particularthat encoding the DBP (E2A), are important.

In addition, in another study, an adenovirus-adenovirus hybrid of humanAd2 and SA7(C8) was shown to be defective for replication, suggestingthat human E1 and monkey E4 are not compatible and that human adenovirusE1 is not sufficient for overcoming the host range restriction, which isconsistent with the above described results where human E1 expressedfrom the cell did not change host range (Alstein et al., JVi, 2: 488(1968); Savitskaya et al., supra). A different adenovirus-adenovirushybrid between Ad2 and SA7(C8) was generated by growth of the twoviruses under selection conditions to prevent Ad2 propagation (Grinenkoet al., Molecular Genetics, Microbiology and Virology, 5:25 (2004)).Growth and selection of the hybrid virus on human cells (HEK-293)yielded a defective virus that had incorporated only the L3 region ofSA7(C8). The authors note that both Ad2 E4 and E2A (encoding the DNAbinding protein) were present and intact in the defective hybrid, andstate that the gene E4 and possibly E2A are involved in thedetermination of species-specific host range, consistent with theearlier conclusions that more than E4 was required for alleviating thehost range restriction. These results showed that only 10% of the Ad2genome could be removed in order for a monkey—human adenovirus hybrid togrow on human cells, leaving 90% of the Ad2 genome to contain host rangedetermining factors. Therefore, this hybrid did not provide furtherdelineation of human adenovirus products required for growth of monkeyadenovirus on human cells. Taken together, these reports showed that E4plays a role in host range determination but other adenovirus genes alsoplay a role. Furthermore, the E4 region is comprised of at least fiveknown protein products, and despite these studies, the component orcomponents of E4 that may have been necessary for the partialalleviation of the host range block were not identified.

Thus, there remains a need for methods that can alleviate, and evenremove, the species-specific block or host range restriction whichprevents a monkey adenovirus from propagating or replicating efficientlyon human cells. There also remains a need for adenoviruses andadenoviral vectors which are capable of circumventing the pre-existingimmunity to adenovirus in humans, The invention provides such methods,adenoviruses and vectors, and methods of using the same,

BRIEF SUMMARY OF THE INVENTION

The invention provides a monkey adenovirus. The monkey adenovirus iscapable of propagation in a cell comprising one or more gene products ofa human adenovirus.

The invention also provides a method for propagating a monkey adenovirusin a cell, wherein the cell comprises a gene product (and/or an encodingnucleic acid sequence) of a human adenovirus. In one embodiment, theinvention provides a cell comprising (a) at least one nucleic acidsequence of the E1 region of a human adenovirus, and (b) at least onenucleic acid sequence of the E4 region of a human adenovirus. In anotherembodiment, there is provided a method of propagating a monkeyadenovirus, which method comprises contacting a cell with the monkeyadenovirus. The cell expresses a gene product encoded by one or both ofthe E1A region and the E1 B region of a human adenovirus, and a geneproduct encoded by a portion of the E4 region consisting essentially ofE4 ORF6 of a human adenovirus, whereby the monkey adenovirus ispropagated in the cell.

The invention also provides a method for propagating a monkey adenovirusin a cell, wherein the monkey adenovirus comprises a nucleic acidsequence encoding a gene product of a human adenovirus. In oneembodiment, the monkey adenovirus comprises (a) at least one nucleicacid sequence of the E1 region of a human adenovirus and (b) at leastone nucleic acid sequence of the E4 region of a human adenovirus. Inanother embodiment, there is provided a method of propagating a monkeyadenovirus, which method comprises contacting a cell with the monkeyadenovirus. In some embodiments, the monkey adenovirus comprises anucleic acid sequence encoding one or more gene products of a humanadenovirus, wherein the one or more gene products comprise a geneproduct encoded by a portion of the E4 region responsible foralleviating or overcoming host replication block in human cells, whichportion consists essentially of E4 ORF6, and whereby the monkeyadenovirus is propagated in the cell. In some embodiments, the one ormore gene products also comprise a gene product encoded by one or bothof the E1 A region and the E1 B region of a human adenovirus.

The invention also provides a monkey adenovirus obtained by thepropagation methods described herein.

In another aspect, the cell in which the monkey adenovirus is propagatedpreferably is a human cell, and the monkey adenovirus in certainembodiments is replication-deficient. The human adenovirus preferably isa species C human adenovirus.

The invention provides a number of advantages over the art, includingmeans for addressing the pre-existing immunity concerns in humans tohuman adenoviruses. The invention also provides greatly improved methodsfor alleviating or overcoming host range restriction or block againstpropagation or replication of a monkey adenovirus in human cells. Theinvention thus enables the substantial growth and use of monkeyadenoviruses for a full range of purposes, including most notably thetreatment and prevention of disease in humans.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph illustrating the classification of the order ofPrimates.

FIG. 2A and FIG. 2B are graphs which illustrate the production of monkeyadenovirus progeny under single-burst conditions. FIG. 2A depictsproduction in human cell lines differing in expressed Ad5 factors(A549=no Ad5 factors, A549+Ad5 E4ORF6=Ad5 E4ORF6 factor, 293−ORF6=Ad5 E1and E4ORF6 factors). FIG. 2B depicts production in human cell lines withAd5 E1 (293 cells), Ad5 E1+Ad5 E4ORF6, or in a monkey cell line (BSC-1).Data are mean +/− standard deviation.

FIG. 3 is a diagram which illustrates a method of constructing monkeyadenoviruses with an expression cassette replacing E1. Monkey adenovirusgenome (SV) with major regions identified (not to scale): the stippledboxes=ITR, Ψ=packaging signal, TATA=E1 a promoter's TATAA box, andcoding regions for E1a, E1 b, pIX, E3 and E4. B is the recipient plasmidwhich comprises an expression cassette comprised of a CMV promoter(arrow), open reading frame (orf), and SV40 polyadenylation signal (SV)linearized between the pIX coding sequence and ITR, bacterial origin ofreplication (Ori) and a gene that encodes Kanamycin (Kan) drugresistance. Homologous recombination (X) between the SV genome andplasmid B results in replacement of the E1 promoter and E1A and E1Bcoding sequences with the CMV-orf expression cassette. Homologousrecombination is generated by transforming recombination competentbacteria BJ5183 with the two DNAs resulting in plasmid C. Bacteria thatare Kan resistant are screened, and plasmid C is identified byrestriction digest and sequencing. Before the viral genome istransfected into 293-ORF6 cells to generate virus particles, plasmid Cis restricted with an endonuclease that recognizes a site (R) outside ofthe viral genome.

DETAILED DESCRIPTION OF THE INVENTION

The invention generally provides methods that alleviate or overcome thespecies-specific block or host range restriction that prevents efficientpropagation or replication of a monkey adenovirus in human cells.

The invention also generally provides a monkey adenovirus, the uses forwhich are accompanied by the advantage of an absence of pre-existingimmunity in human populations to monkey adenoviruses.

In one aspect, the invention provides methods for propagating a monkeyadenovirus in a cell, wherein the cell expresses one or more geneproducts of a human adenovirus, and/or wherein the monkey adenoviruscomprises a nucleic acid sequence encoding a human adenovirus geneproduct.

In a second aspect, the invention provides the monkey adenovirusobtained by such propagation methods.

In a third aspect, the invention provides uses for the monkey adenovirusas vectors.

The invention provides a method of propagating a monkey adenovirusinvolving contacting a cell (e.g., transforming the cell) with theadenovirus. In one embodiment, the cell comprises gene products encodedby one or more of the E1A, E1B, and E4 regions of a human adenovirus. Inanother embodiment, the cell comprises the gene product (and/or itsencoding nucleic acid sequence) responsible for alleviating orovercoming the host replication block of monkey adenoviruses in humancells. In another embodiment, the method comprises contacting a cellwith a monkey adenovirus, wherein the cell expresses a gene productencoded by one or both of the E1A region and the E1 B region of a humanadenovirus, and a gene product encoded by a portion of the E4 regionresponsible for alleviating or overcoming host replication block inhuman cells, which portion consists essentially of E4 ORF6, and wherebythe monkey adenovirus is propagated in the cell. In another embodiment,the method comprises contacting a cell with a monkey adenovirus, whereinthe monkey adenovirus comprises a nucleic acid sequence encoding a humanadenovirus gene product, which may include a gene product encoded by oneor more of the E1A, E1B, and E4 regions of a human adenovirus, and willinclude a gene product encoded by the portion of the E4 regionresponsible for alleviating or overcoming host replication block inhuman cells. In another embodiment, a method of propagating a monkeyadenovirus is provided, which method comprises contacting a cell withthe monkey adenovirus. The monkey adenovirus comprises a nucleic acidsequence encoding one or more gene products of a human adenovirus,wherein the one or more gene products comprise a gene product encoded bya portion of the E4 region responsible for alleviating or overcominghost replication block in human cells, which portion consistsessentially of E4 ORF6, and whereby the monkey adenovirus is propagatedin the cell.

In some embodiments, the cell expresses an E4 region that is responsiblefor alleviating or overcoming the species-specific block. In someembodiments, the E4 region expressed comprises ORF6. In someembodiments, the E4 region expressed consists essentially of ORF6. Insome embodiments, the E4 region expressed consists of ORF6 and no otherORF of the E4 region.

In one embodiment, the cell contacting the monkey adenovirus preferablyexpresses one or more gene products of a species C human adenovirus,which encompasses a number of human adenoviruses, including a preferredhuman serotype 5 adenovirus.

Human cells are preferred for propagating the monkey adenovirus, andpreferred human cells include an HEK-293 cell or a PerC.6 cell.

The monkey adenovirus also may be replication-deficient. Whenreplication-deficient, the adenovirus requires complementation of one ormore of the E1A region, the E1B region, and the E4 region of theadenovirus for propagation. In one embodiment, the monkey adenoviruscomprises a deficiency in the E1 region and a deficiency in at least aportion of the E4 region of the adenoviral genome. In a furtherembodiment, the adenovirus also comprises a deficiency in the E3 regionof the adenoviral genome.

In another embodiment, the monkey adenovirus may comprise a heterologousnucleic acid sequence, including a nucleic acid sequence encoding anantigen. The monkey adenovirus preferably comprises a deletion of the E1region and more preferably also a deletion of at least a portion of theE4 region of the adenovirus, and the heterologous nucleic acid sequenceis inserted into the deleted E1 region or the deleted E4 region of theadenovirus.

The monkey adenovirus may be of various serotypes, known or discoveredin the future, including the following known serotypes 1, 3, 7, 11, 16,18, 19, 20, 27, 33, 38, 39, or combinations thereof.

The term “monkey,” as used herein, refers to both new world and oldworld monkeys, and does not include any member of the family Hominidae(e.g., humans, chimpanzees, gorillas, and orangutans, which are alsoreferred to as the “great apes”). New world monkeys include the familiesCallitrichidae (e.g., marmosets and tamarins), Cebidae (e.g., capuchinsand squirrel monkeys), Aotidae (e.g., night or owl monkeys(douroucoulis)), Pitheeiidae (e.g., titis, sakis and uakaris), andAtelidae (e.g., howler, spider, and woolly monkeys) (see, e.g.,Hershkovitz (ed.), Living New World Monkeys (Platyrrhini), Volume 1,University of Chicago Press (1977)). Old world monkeys include animalsin the family Cercopithecinae, such as, for example, macaques, baboons,and mangabeys (see, e.g., Whitehead, ed., Old World Monkeys, CambridgeUniversity Press (2002)). The term “monkey” also is used synonymouslyherein with the term “simian.” The taxonomy of the order of Primates isillustrated in FIG. 1.

Adenovirus serotypes are differentiated on the basis of neutralizationassays. A serotype is defined as one which either exhibits no or limitedcross-reaction with other types (see, Fauquet et al. (eds.), VirusTaxonomy: The Eighth Report of the International Committee on Taxonomyof Viruses, Academic Press, p. 216 (2005)). The serologicallydistinguishable serotypes (a)so referred to as adenovirus “types”) aregrouped into species. Classically, the species name has reflected thefirst described host. The lack of cross neutralization combined with acalculated phylogenetic distance of more than 10% separates twoserotypes into different species. In addition, species designationdepends on other characteristics that differ between serotypes ofadenovirus, including host range, DNA hybridization, RFLP analysis,percentage of GC in the genome, oncogenicity in rodents, growthcharacteristics, possibility of recombination, number of VA RNA genes,hemagglutination, genetic organization of the E3 region, and host range.Simian adenoviruses isolated from monkeys are more distant from bothhuman adenoviruses and chimpanzee adenoviruses. The chimpanzeeadenoviruses are closely related to common human adenoviruses of speciesB and E, so similar that the chimpanzee adenoviruses are grouped withinthe human species B and E. The limited phylogenic reconstructions forthe simian adenoviruses reveal that the simian adenoviruses are quitedistinct from the common chimpanzee and human adenoviruses (VirusTaxonomy: VIIIth Report of the International Committee on Taxonomy ofViruses (2005)). The phylogeny of adenoviruses that infect primates isdisclosed in, e.g., Roy et al., PLoS Pathog., 5(7): e100050.doi:10.1371/journal.ppat.1000503 (2009).

Various origins, serotypes, or mixtures of serotypes can be used as thesource of the viral genome for the simian adenoviral vector (such asthose described in, e.g., U.S. Pat. Nos. 7,247,472 and 7,491,508). Forinstance, a simian adenovirus can be of serotype 1, 3, 6, 7, 11, 16, 18,19, 20, 27, 33, 38, 39, 48, 49, 50, or any other simian adenoviralserotype. A simian adenovirus can be referred to by using any suitableabbreviation known in the art, such as, for example, SV, SAdV, or SAV.In some embodiments, the simian adenoviral vector is a simian adenoviralvector of serotype 3, 6, 7, 11, 16, 18, 19, 20, 27, 33, 38, or 39. Insome embodiments, the simian adenoviral vector is of serotype 7, 11, 16,18, or 38. In one embodiment, the simian adenoviral vector is ofserotype 7. These simian adenoviruses, isolated from monkeys, have lowsequence homology to human serotype 5 adenovirus, and are more closelyrelated, though quite distinct, from the enteric F and G serotypeadenoviruses. They contain two different fiber genes (long and shortfibers) instead of one fiber gene, which suggests that they may targetthe gut mucosa, similar to gut-tropic human adenoviruses, where they areexpected to stimulate mucosal immune responses. In addition, comparisonsbetween viral hexon proteins suggest that simian adenovirus serotypes 7,11, 16, and 38 are distantly related to human adenoviruses, and arecategorized more closely to gut-tropic adenoviruses (human Ad40, 41, and52) than to other groups.

Wild-type simian adenoviruses of any serotype can be isolated using anysuitable method. For example, simian adenoviruses can be isolated frommonkey biopsy and body secretions, including intestine biopsy, fecalwashes, nose washes, lung washes, and other body secretions usingstandard methods in the art. Wild-type simian adenoviruses also areavailable from commercial sources, such as the American Type CultureCollection (ATCC, Manassas, Va.).

In softie embodiments, the simian adenoviruses are from baboon (e.g.,ATCC-VR 275) or Rhesus or African Green monkeys (e.g., ATCC-VR 196,ATCC-VR 201, ATCC-VR 209, ATCC-VR 353, ATCC-VR 355, ATCC-VR 541, ATCC-VR941, ATCC-VR 942, and ATCC-VR 943).

The invention provides improved monkey adenovirus replication in humancells with preferably complete alleviation of host range block in mostcases. The data presented herein confirms that monkey adenoviruses donot grow on human cells and demonstrates equal or even superior growthof monkey adenoviruses on human cells with human adenovirus componentscompared to monkey cells (see Example 1). Example 1 shows the highproductivity of monkey adenoviral progeny on a human cell line withminimal human adenovirus components, comparable or even higher than onmonkey cells, in contrast to the 40-fold deficit reported in Savitskayaet al., supra. In some embodiments, the host range restriction can beremoved by propagating the monkey virus on human embryonic kidney cellline 293 (HEK-293) along with human adenovirus E4 ORF6 protein (34K)expressed during adenovirus infection. Surprisingly, the expression ofthe 34k protein in the HEK-293 cell in Example 1 was too low to bedetected, reminiscent to that reported previously for VK-10-9 cells(Krougliak and Graham, Hum. Gene Ther., 6: 1575 (1995)). Thus it wasunexpected that the human cell line in Example 1 was equally permissivefor monkey adenoviruses as are monkey cells for monkey adenoviruses(data not shown).

Thus, in accordance with the invention, in order to overcome the hostrange restriction of monkey adenoviruses to grow on human cells, asubset or portion of human adenovirus E4 must be expressed during viralreplication in the cell instead of the whole E4 region, as the functionof E4 that overcomes the replication block in human cells lies withinORF6. It is possible that the reason for the failure of VK-10-9 cells tofully support monkey adenovirus growth was the presence of inhibitoryfunctions in the human E4 sequences inserted into the cell. Thus, thehost range determinant does not include all of E4, and does not includeE2A. Rather, the host range determinant is E4 ORF6, and apparently notone of the other factors singly or in combination encoded within E4. Inparticular, in light of the discovery of E4 ORF6 being sufficient, thedata in Savitskaya et al., supra, could he re-interpreted in that lessof E4 was required instead of more, and that there may be components inhuman adenovirus E4, included in VK-10-9 cells, which inhibit growth ofmonkey adenovirus in human cells.

The identification of the human adenovirus components which permitreplication of monkey adenovirus on human cells has many advantages,such as, but not limited to, feasible manufacture of products based onmonkey adenovirus. In addition, the ability to reduce the components ofE4 required to alleviate the host range block to monkey adenoviruses onhuman cells has clear advantages compared to requiring all of E4. Thesimplification of the E4 requirement allows for easier manipulation ofthe DNA sequences and proper regulation of expression of the E4sequences. This allows for easier design of systems to allow propagationof monkey viruses on human cells. For example, the subset of humanadenovirus E4 sequences can be included in a monkey adenovirus genome,integrated into the genome of a cell or exist extra chromosomally asneither part of the human cell nor the monkey adenovirus. Working withonly a subset of E4 sequences that comprise E4 ORF6 allows for easierand more reliable regulation of expression of these sequences. Thisenhanced control leads to higher yields of monkey adenoviruses whichwill allow for reduction in cost of goods and expand the commercial andscientific applications that monkey adenoviruses can be used for.

Another advantage of the identification of the human adenoviruscomponents sufficient for allowing replication of monkey adenovirus onhuman cells is the ability to generate conditionally replicatingadenoviruses (CRADs). For example, the inclusion of human adenovirus E1and E4 ORF6 sequences in the monkey adenovirus, under expression controlelements that are specific to a given disease, syndrome, condition,tissue, or cell type, allow for replication of the monkey virus in acontrolled fashion only where desired. Applications for CRADs arenumerous and include, for example, lysis of tumor cells, expression of atherapeutic gene only under conditions of viral vector replication, andlimited-replication vaccines.

Another advantage of the identification of the human adenoviruscomponents sufficient for significant replication of monkey adenoviruson human cells is the ability to generate adenovirus gene transfervectors where a transgene expression cassette is incorporated into themonkey adenovirus genome. Adenovirus vectors derived from monkeyadenoviruses propagated on a human cell line-human adenovirus systemhave the following advantages: (1) absence of pre-existing immunity inhuman populations to the monkey adenoviruses, (2) species-specific blockto replication for enhanced safety to human populations, and (3) avoidsthe risk of adventitious xenogeneic pathogens from manufacturing on anon-human cell line. For example, the monkey polyoma virus SV40 wasfound to contaminate batches of human vaccine product manufactured onmonkey cells.

Complementing cell lines for producing the simian adenoviral vectorinclude, but are not limited to, 293 cells (described in, e.g., Grahamet al., J. Gen. Virol., 36: 59-72 (1977)), PER.C6 cells (described in,e.g., International Patent Application Publication WO 97/00326, and U.S.Pat. Nos. 5,994,128 and 6,033,908), and 293-ORF6 cells (described in,e.g., international Patent Application Publication WO 95/34671 andBrough et al., J. Virol., 71: 9206-9213 (1997)). Additionalcomplementing cells are described in, for example, U.S. Pat. Nos.6,677,156 and 6,682,929, and International Patent ApplicationPublication WO 03/20879. In some instances, the cellular genome need notcomprise nucleic acid sequences, the gene products of which complementfor all of the deficiencies of a replication-deficient adenoviralvector. One or more replication-essential gene functions lacking in areplication-deficient adenoviral vector can be supplied by a helpervirus, e.g., an adenoviral vector that supplies in trans one or moreessential gene functions required for replication of the desiredadenoviral vector. Helper virus is often engineered to prevent packagingof infectious helper virus. For example, one or morereplication-essential gene functions of the E1 region of the adenoviralgenome are provided by the complementing cell, while one or morereplication-essential gene functions of the E4 region of the adenoviralgenome are provided by a helper virus.

Ideally, a replication-deficient simian adenoviral vector is present ina composition, e.g., a pharmaceutical composition, substantially free ofreplication-competent adenovirus (RCA) contamination (e.g., thecomposition comprises less than about 1% of replication-competentadenovirus on the basis of the total adenoviruses in the composition).Most desirably, the composition is RCA-free. Adenoviral vectorcompositions and stocks that are RCA-free are described in U.S. Pat. No.5,944,106, U.S. Patent Application Publication 2002/0110545 A1, andInternational Patent Application WO 95/34671.

If the simian adenoviral vector is not replication-deficient, ideallythe simian adenoviral vector is manipulated to limit replication of thevector to within a target tissue. For example, the simian adenoviralvector can be a conditionally-replicating adenoviral vector, which isengineered to replicate under conditions pre-determined by thepractitioner. For example, replication-essential gene functions, e.g.,gene functions encoded by the adenoviral early regions, can be operablylinked to an inducible, repressible, or tissue-specific transcriptioncontrol sequence, e.g., promoter. In this embodiment, replicationrequires the presence or absence of specific factors that interact withthe transcription control sequence. In the treatment of viralinfections, for example, it can be advantageous to control adenoviralvector replication in, fur instance, lymph nodes, to obtain continualantigen production and control immune cell production.Conditionally-replicating adenoviral vectors are described further inU.S. Pat. No. 5,998,205.

One of the utilities of a monkey adenovirus is to deliver proteins orparts of proteins to a cell. One method of delivering the proteins is totether them to one of the coat proteins of the viral capsid. The capsidcan be modified to facilitate this. There are numerous examples of theviral capsid being modified to tether non-adenovirus materials to thecapsid. Some of the modifications are proteinacious character, whileothers are not. Examples of substances which can be linked to theadenovirus capsid include antibodies, receptors, PEG, and cross linkingchemicals. The viral capsid genes can also be genetically modified toinclude a foreign gene or portion thereof, so that the foreign geneproduct is part of the viral capsid protein which becomes part of theviral particle. The effect of the protein can be exerted on the cellwith or without it being internalized. If it is not internalized itcould activate or inactivate a cell pathway leading to a desiredoutcome. In addition, if the monkey virus is internalized the proteincould elicit an effect on the cell. The protein could also stimulate animmune response potentially to the protein itself. The capsid proteinsfiber, hexon, pIX, and penton have all been shown to be able to bemodified to include non-adenoviral proteins or portions thereof and ornon-proteinacious materials.

Modification of the viral capsid can have additional benefits. Thevirus's natural tropism can be changed. The virus could be redirected toa new receptor, its interaction with its normal receptor can beabrogated, or its interaction with its normal receptor can be enhanced.Re-directing the monkey adenovirus can increase the desired activity ofthe virus by directing it to the desired cell type, or it can help avoida cell type that would yield a non-desirable outcome. The modificationcan also lead to evasion of the immune system. Multiple capsid proteinscan be modified simultaneously.

The coat protein of the adenovirus can be manipulated to alter thebinding specificity or recognition of the adenovirus for a receptor on apotential host cell. For adenovirus, such manipulations can includedeletion of regions of adenovirus coat proteins (e.g., fiber, penton, orhexon), insertions of various native or non-native ligands into portionsof a coat protein, and the like. Manipulation of the coat protein canbroaden the range of cells infected by the adenovirus or enabletargeting of the adenovirus to a specific cell type.

The simian adenoviral vector can be manipulated to alter the bindingspecificity or recognition of the adenovirus for a receptor on apotential host cell. For adenovirus, such manipulations can includedeletion of regions of adenovirus coat proteins (e.g., fiber, penton, orhexon), insertions of various native or non-native ligands into portionsof a coat protein, and the like. Manipulation of the coat protein canbroaden the range of cells infected by the simian adenoviral vector orenable targeting of the simian adenoviral vector to a specific celltype. It can also avoid interaction with proteins found in the blood,such as coagulation factor X (FX), which can affect the adenoviralvector biology. Modification of hexon is the preferable method ofavoiding the interaction with FX.

Any suitable technique for altering native binding to a host cell, suchas native binding of the fiber protein to its cellular receptor, can beemployed. For example, differing fiber lengths can be exploited toablate native binding to cells. This optionally can be accomplished viathe addition of a binding sequence to the penton base or fiber knob.This addition of a binding sequence can be done either directly orindirectly via a bispecific or multispecific binding sequence. In analternative embodiment, the adenoviral fiber protein can be modified toreduce the number of amino acids in the fiber shaft, thereby creating a“short-shafted” fiber (as described in, for example, U.S. Pat. No.5,962,311). Use of an adenovirus comprising a short-shafted adenoviralfiber gene reduces the level or efficiency of adenoviral fiber bindingto its cell-surface receptor and increases adenoviral penton basebinding to its cell-surface receptor, thereby increasing the specificityof binding of the adenovirus to a given cell. Alternatively, use of asimian adenoviral vector comprising a short-shafted fiber enablestargeting of the simian adenoviral vector to a desired cell-surfacereceptor by the introduction of a normative amino acid sequence eitherinto the penton base or the fiber knob.

In yet another embodiment, the nucleic acid residues encoding amino acidresidues associated with native substrate binding can be changed,supplemented, or deleted (see, e.g., International Patent ApplicationPublication WO 00/15823, Einfeld et al., J. Virol., 75(23): 11284-11291(2001), and van Beusechem et al., J. Virol., 76(6): 2753-2762 (2002))such that the simian adenoviral vector incorporating the mutated nucleicacid residues (or having the fiber protein encoded thereby) is less ableto bind its native substrate.

Any suitable amino acid residue(s) of a fiber protein that mediates orassists in the interaction between the knob and the native cellularreceptor can be mutated or removed, so long as the fiber protein is ableto trimerize. Similarly, amino acids can be added to the fiber knob aslong as the fiber protein retains the ability to trimerize. Suitableresidues include amino acids within the exposed loops of the fiber knobdomain, such as, for example, the AB loop, the DE loop, the FG loop, andthe HI loop.

Any suitable amino acid residue(s) of a penton base protein thatmediates or assists in the interaction between the penton base andintegrins can be mutated or removed. Suitable residues include, forexample, an RGD amino acid sequence motif located in the hypervariableregion of the simian adenovirus penton base protein. The native integrinbinding sites on the penton base protein also can be disrupted bymodifying the nucleic acid sequence encoding the native RGD motif suchthat the native ROD amino acid sequence is conformationally inaccessiblefor binding to an integrin receptor, such as by inserting a DNA sequenceinto or adjacent to the nucleic acid sequence encoding the adenoviralpenton base protein.

The simian adenoviral vector can comprise a fiber protein and a pentonbase protein that do not hind to their respective native cellularbinding sites. Alternatively, the simian adenoviral vector comprisesfiber protein and a penton base protein that bind to their respectivenative cellular binding sites, but with less affinity than thecorresponding wild-type coat proteins. The simian adenoviral vectorexhibits reduced binding to native cellular binding sites if a modifiedadenoviral fiber protein and penton base protein binds to theirrespective native cellular binding sites with at least about 5-fold,10-fold, 20-fold, 30-fold, 50-fold, or 100-fold less affinity than anon-modified adenoviral fiber protein and penton base protein of thesame serotype.

The simian adenoviral vector also can comprise a chimeric coat proteincomprising a non-native amino acid sequence that binds a substrate(i.e., a ligand), such as a cellular receptor other than a nativecellular receptor. The non-native amino acid sequence of the chimericadenoviral coat protein allows the simian adenoviral vector comprisingthe chimeric coat protein to bind and, desirably, infect host cells notnaturally infected by a corresponding adenovirus without the non-nativeamino acid sequence (i.e., host cells not infected by the correspondingwild-type adenovirus), to bind to host cells naturally infected by thecorresponding wild-type adenovirus with greater affinity than thecorresponding adenovirus without the non-native amino acid sequence, orto bind to particular target cells with greater affinity than non-targetcells. A “non-native” amino acid sequence can comprise an amino acidsequence not naturally present in the adenoviral coat protein or anamino acid sequence found in the adenoviral coat but located in anon-native position within the capsid. By “preferentially binds” ismeant that the non-native amino acid sequence binds a receptor, such as,for instance, αbβ3 integrin, with at least about 3-fold greater affinity(e.g., at least about 5-fold, 10-fold, 15-fold, 20-fold, 25-fold,35-fold, 45-fold, or 50-fold greater affinity) than the non-nativeligand binds a different receptor, such as, for instance, αvβ1 integrin.

The simian adenoviral vector can comprise a chimeric coat proteincomprising a non-native amino acid sequence that confers to the chimericcoat protein the ability to bind to an immune cell more efficiently thana wild-type adenoviral coat protein. In particular, the simianadenoviral vector can comprise a chimeric adenoviral fiber proteincomprising a non-native amino acid sequence which facilitates uptake ofthe simian adenoviral vector by immune cells, preferably antigenpresenting cells, such as dendritic cells, monocytes, and macrophages.In a preferred embodiment, the simian adenoviral vector comprises achimeric fiber protein comprising an amino acid sequence (e.g.,anon-native amino acid sequence) comprising an RGD motif, whichincreases transduction efficiency of the simian adenoviral vector intodendritic cells. The RGD-motif, or any non-native amino acid sequence,preferably is inserted into the adenoviral fiber knob region, ideally inan exposed loop of the adenoviral knob, such as the HI loop. Anon-native amino acid sequence also can be appended to the C-terminus ofthe adenoviral fiber protein, optionally via a spacer sequence. Thespacer sequence preferably comprises between one and two-hundred aminoacids, and can (but need not) have an intended function.

In another embodiment, the simian adenoviral vector can comprise achimeric virus coat protein that is not selective for a specific type ofeukaryotic cell. The chimeric coat protein differs from a wild-type coatprotein by an insertion of a non-native amino acid sequence into or inplace of an internal coat protein sequence, or attachment of anon-native amino acid sequence to the N- or C-terminus of the coatprotein. For example, a ligand comprising about five to about ninelysine residues (preferably seven lysine residues) can be attached tothe C-terminus of the adenoviral fiber protein via a non-functionalspacer sequence. In this embodiment, the chimeric virus coat proteinefficiently binds to a broader range of eukaryotic cells than awild-type virus coat, such as described in U.S. Pat. No. 6,465,253 andInternational Patent Application Publication WO 97/20051.

The ability of the simian adenoviral vector to recognize a potentialhost cell can be modulated without genetic manipulation of the coatprotein, i.e., through use of a bi-specific molecule. For instance,complexing an adenovirus with a bispecific molecule comprising a pentonbase-binding domain and a domain that selectively binds a particularcell surface binding site enables the targeting of the simian adenoviralvector to a particular cell type. Likewise, an antigen can be conjugatedto the surface of the adenoviral particle through non-genetic means.

A non-native amino acid sequence can be conjugated to any of theadenoviral coat proteins to form a chimeric adenoviral coat protein.Therefore, for example, a non-native amino acid sequence can beconjugated to, inserted into, or attached to a fiber protein, a pentonbase protein, a hexon protein, protein IX, VI, or IIIa, etc. Methods foremploying such proteins are well known in the art (see, e.g., U.S. Pat.Nos. 5,543,328; 5,559,099; 5,712,136; 5,731,190; 5,756,086; 5,770,442;5,846,782; 5,962,311; 5,965,541; 5,846,782; 6,057,155; 6,127,525;6,153,435; 6,329,190; 6,455,314; 6,465,253; 6,576,456; 6,649,407;6,740,525; and 6,951,755, and International Patent ApplicationPublications WO 96/07734, WO 96/26281, WO 97/20051, WO 98/07877, WO98/07865, WO 98/40509, WO 98/54346, WO 00/15823, WO 01/58940, and WO01/92549). The chimeric adenoviral coat protein can be generated usingstandard recombinant DNA techniques known in the art. Preferably, thenucleic acid sequence encoding the chimeric adenoviral coat protein islocated within the adenoviral genome and is operably linked to apromoter that regulates expression of the coat protein in a wild-typeadenovirus. Alternatively, the nucleic acid sequence encoding thechimeric adenoviral coat protein is located within the adenoviral genomeand is part of an expression cassette which comprises genetic elementsrequired for efficient expression of the chimeric coat protein.

The coat protein portion of the chimeric adenovirus coat protein can bea full-length adenoviral coat protein to which the non-native amino acidsequence is appended, or it can be truncated, e.g., internally or at theC- and/or N-terminus. However modified (including the presence of thenon-native amino acid), the chimeric coat protein preferably is able toincorporate into an adenoviral capsid. Where the non-native amino acidsequence is attached to the fiber protein, preferably it does notdisturb the interaction between viral proteins or fiber monomers. Thus,the non-native amino acid sequence preferably is not itself anoligomerization domain, as such can adversely interact with thetrimerization domain of the adenovirus fiber. Preferably the non-nativeamino acid sequence is added to the virion protein, and is incorporatedin such a manner as to be readily exposed to a substrate, cellsurface-receptor, or immune cell (e.g., at the N- or C-terminus of theadenoviral protein, attached to a residue facing a substrate, positionedon a peptide spacer, etc.) to maximally expose the non-native amino acidsequence. Ideally, the non-native amino acid sequence is incorporatedinto an adenoviral fiber protein at the C-terminus of the fiber protein(and attached via a spacer) or incorporated into an exposed loop (e.g.,the HI loop) of the fiber to create a chimeric coat protein. Where thenon-native amino acid sequence is attached to or replaces a portion ofthe penton base, preferably it is within the hypervariable regions toensure that it contacts the substrate, cell surface receptor, or immunecell. Where the non-native amino acid sequence is attached to the hexon,preferably it is within a hypervariable region (Crawford-Miksza et al.,J. Virol., 70(3): 1836-44 (1996)). Where the non-native amino acid isattached to or replaces a portion of pIX, preferably it is within theC-terminus of pIX. Use of a spacer sequence to extend the non-nativeamino acid sequence away from the surface of the adenoviral particle canbe advantageous in that the non-native amino acid sequence can be moreavailable for binding to a receptor, and any steric interactions betweenthe non-native amino acid sequence and the adenoviral fiber monomers canbe reduced.

In other embodiments (e.g., to facilitate purification or propagationwithin a specific engineered cell type), a non-native amino acid (e.g.,ligand) can hind a compound other than a cell-surface protein. Thus, theligand can bind blood- and/or lymph-borne proteins (e.g., albumin),synthetic peptide sequences such as polyamino acids (e.g., polylysine,polyhistidine, etc.), artificial peptide sequences (e.g., FLAG), and RODpeptide fragments (Pasqualini et al., J. Cell. Biol., 130: 1189 (1995)).A ligand can even bind non-peptide substrates, such as plastic (e.g.,Adey et al., Gene, 156: 27 (1995)), biotin (Saggio et al., Biochem. J.,293: 613 (1993)), a DNA sequence (Cheng et al., Gene, 171: 1 (1996), andKrook et al., Biochem. Biophys., Res. Commun., 204: 849 (1994)),streptavidin (Geibel et al., Biochemistry, 34: 15430 (1995), and Katz,Biochemistry, 34: 15421 (1995)), nitrostreptavidin (Balass et al., Anal.Biochem., 243: 264 (1996)), heparin (Wickham et al., Nature Biotechnol.,14: 1570-73 (1996)), and other substrates.

Disruption of native binding of adenoviral coat proteins to a cellsurface receptor can also render it less able to interact with theinnate or acquired host immune system. Adenoviral vector administrationinduces inflammation and activates both innate and acquired immunemechanisms. Adenoviral vectors activate antigen-specific (e.g., T-celldependent) immune responses, which limit the duration of transgeneexpression following an initial administration of the vector. Inaddition, exposure to adenoviral vectors stimulates production ofneutralizing antibodies by B cells, which can preclude gene expressionfrom subsequent doses of adenoviral vector (Wilson & Kay, Nat, Med.,3(9): 887-889 (1995)), Indeed, the effectiveness of repeatedadministration of the vector can be severely limited by host immunity.In addition to stimulation of humoral immunity, cell-mediated immunefunctions are responsible for clearance of the virus from the body.Rapid clearance of the virus is attributed to innate immune mechanisms(see, e.g., Worgall et al., Human Gene Therapy, 8: 37-44 (1997)), andlikely involves Kupffer cells found within the liver. Thus, by ablatingnative binding of an adenovirus fiber protein and penton base protein,immune system recognition of an adenoviral vector is diminished, therebyincreasing vector tolerance by the host.

A method for evading pre-existing host immunity to adenovirus involvesmodifying an adenoviral coat protein such that it exhibits reducedrecognition by the host immune system. The modified coat proteinpreferably is a penton, fiber, or hexon protein. Most preferably, themodified coat protein is a hexon protein. The coat protein can bemodified in any suitable manner, but is preferably modified bygenerating diversity in the coat protein. Preferably, such coat proteinvariants are not recognized by pre-existing host adenovirus-specificneutralizing antibodies. Diversity can be generated using any suitablemethod known in the art, including, for example, directed evolution(i.e., polynucleotide shuffling) and error-prone PCR (see, e.g.,Cadwell, PCR Meth. Appl., 2: 28-33 (1991), Leung et al., Technique, 1:11-15 (1989), and Pritchard et al., J Theoretical Biol., 234: 497-509(2005)). Immune avoidance also includes pegylation and the like.

An adenoviral coat protein also can be modified to evade pre-existinghost immunity by deleting a region of a coat protein and replacing itwith a corresponding region from the coat protein of another adenovirusserotype, particularly a serotype which is less immunogenic in humans.In this regard, amino acid sequences within the fiber protein, thepenton base protein, and/or hexon protein can be removed and replacedwith corresponding sequences from a different adenovirus serotype. Thus,for example, when the fiber protein is modified to evade pre-existinghost immunity, amino acid residues from the knob region of a simianadenovirus fiber protein can be deleted and replaced with correspondingamino acid residues from a simian adenovirus of a different serotype,such as those serotypes described herein. Likewise, when the penton baseprotein is modified to evade pre-existing host immunity, amino acidresidues within the hypervariable region of a simian adenovirus pentonbase protein can be deleted and replaced with corresponding amino acidresidues from a simian adenovirus of a different serotype, such as thoseserotypes described herein. Preferably, the hexon protein of the simianadenoviral vector is modified in this manner to evade pre-existing hostimmunity. In this respect, amino acid residues within one or more of thehypervariable regions, which occur in loops of the hexon protein, areremoved and replaced with corresponding amino acid residues from asimian adenovirus of a different serotype. An entire loop region can beremoved from the hexon protein and replaced with the corresponding loopregion of another simian adenovirus serotype. Alternatively, portions ofa loop region can be removed from the simian adenoviral vector hexonprotein and replaced with the corresponding portion of a hexon loop ofanother adenovirus serotype (simian or human), One or more hexon loops,or portions thereof, of a simian adenoviral vector can be removed andreplaced with the corresponding sequences from any other adenovirusserotype (simian or human), such as those described herein. Methods ofmodifying hexon proteins are disclosed in, for example, Rug: et al., J.Virol., 77:9553-9566 (2003), and U.S. Pat. No. 6,127,525. Thehypervariable regions of a hexon protein also can be replaced withrandom peptide sequences, or peptide sequences derived from adisease-causing pathogen (e.g., HIV).

Modifications to adenovirus coat proteins are described in, for example,U.S. Pat. Nos. 5,543,328; 5,559,099; 5,712,136; 5,731,190; 5,756,086;5,770,442; 5,846,782; 5,871,727; 5,885,808; 5,922,315; 5,962,311;5,965,541; 6,057,155; 6,127,525; 6,153,435; 6,329,190; 6,455,314;6,465,253; 6,576,456; 6,649,407; 6,740,525; and 6,951,755; andInternational Patent Applications WO 96/07734, WO 96/26281, WO 97/20051,WO 98/07865, WO 98/07877, WO 98/40509, WO 98/54346, WO 00/15823, WO01/58940, and WO 01/92549.

Monkey adenovirus can also be used to deliver genetic material to acell. The genetic material is typically DNA. Any DNA that is inserted inthe monkey virus genome is referred to herein as a heterologous nucleicacid sequence or “hDNA.” There are a number of functions the hDNA canhave that are known in the art. The hDNA can have regulatory properties.Some of the more common elements regulate transcription such aspromoters, enhancers, transcriptional terminators, splicing elements,matrix attachment regulatory elements, transcriptional insulators, andpoly adenylation sequences to name a few. If RNA is generated from thehDNA it can have a function. Some of the functions are regulatory innature as shown with siRNA, shRNA, microRNA, anti sense RNA, VA RNA. TheRNA can also encode a polypeptide such as a protein. The protein can benative or modified in any fashion that is known in the art. There aremany ways to regulate levels of RNA, translation and protein stabilitythat are known in the art.

There are numerous functions the regulatory RNAs and polypeptides canhave.

The heterologous nucleic acid sequence preferably encodes an antigen ofa pathogen. The pathogen can be a virus, such as respiratory syncitialvirus (RSV), human immunodeficiency virus (HIV), foot-and-mouth disease(FMDV), herpes simplex virus (HSV), hepatitis C virus (HCV), ebolavirus, or Marburg virus. The pathogen also can be a parasite, such as,for example, a Plasmodium parasite, which causes malaria (e.g.,Plasmodium falciparum). Alternatively, the heterologous nucleic acidsequence can encode, for example, an atonal homolog protein (e.g., HATH1or MATH1), TNF-α, or pigment epithelium-derived factor (PEDF).

The adenoviral vector of the invention can be replication competent, Forexample, the adenoviral vector can have a mutation (e.g., a deletion, aninsertion, or a substitution) in the adenoviral genome that does notinhibit viral replication in host cells. Preferably, however, theadenoviral vector is replication-deficient. By “replication-deficient”is meant that the adenoviral vector comprises an adenoviral genome thatlacks at least one replication-essential gene function (i.e., such thatthe adenoviral vector does not replicate in typical host cells,especially those in a human patient that could be infected by theadenoviral vector in the course of the inventive method). A deficiencyin a gene, gene function, or gene or genomic region, as used herein, isdefined as a deletion of sufficient genetic material of the viral genometo impair or obliterate the function of the gene whose nucleic acidsequence was deleted in whole or in part. While deletion of geneticmaterial is preferred, mutation of genetic material by addition orsubstitution also is appropriate for disrupting gene function.Replication-essential gene functions are those gene functions that arerequired for replication (e.g., propagation) and are encoded by, forexample, the adenoviral early regions (e.g., the E1, E2, and E4regions), late regions (e.g., the L1-L5 regions), genes involved inviral packaging (e.g., the IVa2 gene), and virus-associated RNAs (e.g.,VA-RNA1 and/or VA-RNA-2). More preferably, the replication-deficientadenoviral vector comprises an adenoviral genome deficient in at leastone replication-essential gene function of one or more regions of theadenoviral genome. Preferably, the adenoviral vector is deficient in atleast one gene function of the E1A region, the E1B region, or the E4region of the adenoviral genome required for viral replication (denotedan E1-deficient or E4-deficient adenoviral vector). In addition to adeficiency in the E1 region, the recombinant adenovirus also can have amutation in the major late promoter (MLP), as discussed in InternationalPatent Application WO 00/00628. In some embodiments, the adenoviralvector is deficient in at least one replication-essential gene function(desirably all replication-essential gene functions) of the E1 regionand at least one gene function of the nonessential E3 region (e.g., anXba I deletion of the E3 region) (denoted an E1/E3-deficient adenoviralvector). With respect to the E1 region, the adenoviral vector can bedeficient in all or part of the E1A region and all or part of the E1 Bregion. To illustrate but not limit this embodiment, a serotype 35adenoviral vector can comprise an E1 deletion of nucleotides 570 to3484. When the adenoviral vector is deficient in at least onereplication-essential gene function in only one region of the adenoviralgenome (e.g., an E1- or E1/E3-deficient adenoviral vector), theadenoviral vector is referred to as “singly replication-deficient.”

The simian adenoviral vector of the invention can be “multiplyreplication-deficient,” meaning that the adenoviral vector is deficientin one or more replication-essential gene functions in each of two ormore regions of the adenoviral genome. For example, the aforementionedE1-deficient or E1/E3-deficient adenoviral vector can be furtherdeficient in at least one replication-essential gene function of the E4region (denoted an E1/E4- or E1/E3/E4-deficient adenoviral vector),and/or the E2 region (denoted an E1/E2- or E1/E2/E3-deficient adenoviralvector), preferably the E2A region (denoted an E1/E2A- orE1/E2A/E3-deficient adenoviral vector).

By removing all or part of, for example, the E1, E3, and E4 regions ofthe adenoviral genome, the resulting adenoviral vector is able to acceptinserts of exogenous nucleic acid sequences while retaining the abilityto be packaged into adenoviral capsids. The nucleic acid sequence can bepositioned in the E1 region, the E3 region, or the E4 region of theadenoviral genome. Indeed, the nucleic acid sequence can be insertedanywhere in the adenoviral genome so long as the position does notprevent expression of the nucleic acid sequence or interfere withpackaging of the adenoviral vector. The adenoviral vector also cancomprise multiple (i.e., two or more) nucleic acid sequences encodingthe same antigen. Alternatively, the adenoviral vector can comprisemultiple nucleic acid sequences encoding two or more different antigens.Each nucleic acid sequence can be operably linked to the same promoter,or to different promoters depending on the expression profile desired bythe practitioner, and can be inserted in the same region of theadenoviral genome (e.g., the E4 region) or in different regions of theadenoviral genome.

The simian adenoviral vector, when multiply replication-deficient,especially in replication-essential gene functions of the E1 and E4regions, preferably includes a spacer sequence to provide viral growthin a complementing cell line similar to that achieved by singlyreplication-deficient adenoviral vectors, particularly an E1-deficientadenoviral vector. The spacer sequence can contain any nucleotidesequence or sequences which are of a desired length, such as sequencesat least about 15 base pairs (e.g., between about 15 base pairs andabout 12,000 base pairs), preferably about 100 base pairs to about10,000 base pairs, more preferably about 500 base pairs to about 8,000base pairs, even more preferably about 1,500 base pairs to about 6,000base pairs, and most preferably about 2,000 to about 3,000 base pairs inlength. The spacer element sequence can be coding or non-coding andnative or non-native with respect to the adenoviral genome, but does notrestore the replication-essential function to the deficient region. Thespacer element preferably is located in the E4 region of the adenoviralgenome. The use of a spacer in an adenoviral vector is described in U.S.Pat. No. 5,851,806.

It has been observed that an at least E4-deficient adenoviral vectorexpresses a transgene at high levels for a limited amount of time invivo and that persistence of expression of a transgene in an at leastE4-deficient adenoviral vector can be modulated through the action of atrans-acting factor, such as HSV ICP0, Ad pTP, CMV-IE2, CMV-IE86, HIVtat, HTLV-tax, HBV-X, AAV Rep 78, the cellular factor from the U205osteosarcoma cell line that functions like HSV ICP0, or the cellularfactor in PC12 cells that is induced by nerve growth factor, amongothers, as described in for example, U.S. Pat. No. 6,225,113, U.S.Patent Application Publication 2002/0031823 A 1, and InternationalPatent Application WO 00/34496. In view of the above, a multiplydeficient adenoviral vector (e.g., the at least E4-deficient adenoviralvector) or a second expression vector desirably comprises a nucleic acidsequence encoding a trans-acting factor that modulates the persistenceof expression of the nucleic acid sequence. Persistent expression ofantigenic DNA can be desired when generating immune tolerance.

The simian adenoviral vector can be deficient in replication-essentialgene functions of only the early regions of the adenoviral genome, onlythe late regions of the adenoviral genome, and both the early and lateregions of the adenoviral genome. The simian adenoviral vector also canhave essentially the entire adenoviral genome removed, in which case itis preferred that at least either the viral inverted terminal repeats(ITRs) and one or more promoters or the viral ITRs and a packagingsignal are left intact (i.e., an adenoviral amplicon). In oneembodiment, the simian adenoviral vector of the invention may comprisean adenoviral genome that lacks native nucleic acid sequences whichencode adenoviral proteins. Adenoviral genomic elements required forreplication and packaging of the adenoviral genome into adenoviralcapsid proteins can be retained. Minimal adenoviral vectors lackingadenoviral protein coding sequences are termed “helper-dependent”adenoviral vectors, and often require complementation by helperadenovirus for efficient propagation. Suitable replication-deficientadenoviral vectors, including multiply replication-deficient adenoviralvectors, are disclosed in U.S. Pat. Nos. 5,837,511; 5,851,806;5,994,106; 6,127,175; and 6,482,616; U.S. Patent ApplicationPublications 2001/0043922 A1, 2002/0004040 A 1, 2002/0031831 A1, and2002/0110545 A1, and International Patent Applications WO 94/28152, WO95/02697, WO 95/16772, WO 95/34671, WO 96/22378, WO 97/12986, WO97/21826, and WO 03/022311.

If the adenoviral vector is not replication-deficient, ideally theadenoviral vector is manipulated to limit replication of the vector towithin a target tissue. For example, the adenoviral vector can be aconditionally-replicating adenoviral vector, which is engineered toreplicate under conditions pre-determined by the practitioner. Forexample, replication-essential gene functions, e.g., gene functionsencoded by the adenoviral early regions, can be operably linked to aninducible, repressible, or tissue-specific transcription controlsequence, e.g., promoter. In this embodiment, replication requires thepresence or absence of specific factors that interact with thetranscription control sequence. In autoimmune disease treatment, it canbe advantageous to control adenoviral vector replication in, forinstance, lymph nodes, to obtain continual antigen production andcontrol immune cell production. Conditionally-replicating adenoviralvectors are described further in U.S. Pat. No. 5,998,205.

It can be advantageous to add large amounts of IONA in the monkeyadenovirus. This can occur for example if large regulatory sequences areneeded, multiple transcriptional units are needed or the transcript islarge. Since the upper packaging size limit of an adenovirus isapproximately 105% of its wild type genome, viruses with larger genomesare difficult to make and are often times unstable.

To overcome this packaging and stability limitation viral DNA sequencescan be deleted to accommodate the large amounts of hDNA. There are atleast three viral regions (i.e., the E 1, E3 and E4 regions) that can beremoved from the virus and still be able to generate infectious viralparticles. Each of these viral regions is composed of at least onepromoter and polyadenylation signal, which encodes fur multipletranscripts and proteins. All or portions of these regions can bedeleted from the viral genome. It is known in the art that hDNA has beeninserted or used to replace each of these regions. These regions can bemodified one at a time or in combination to yield an E1, E3 and E4modified virus. This further expands the flexibility and therefore theutility of the monkey virus.

The removal of the E1, E3 and E4 regions from the virus has additionalbenefits for the use of monkey adenoviruses. These regions are known toencode multiple regulatory proteins that can alter the host celldirectly or stimulate the expression of additional viral proteins, TheE1 and E4 regions in particular are known to encode oncogenes. In manyapplications it is preferred that the virus's genes are not expressed.Some of the benefits of less viral gene expression is improved activityfrom the added hDNA such as gene expression, avoidance of immunesurveillance when the virus is administered to an animal and increasedvirus dose that can be administered. The ability to increase dosage andimprove hDNA gene regulation expands the applications and thereforeutility of the monkey virus. Therefore deleting viral DNA willsimultaneously expand the amount of hDNA the virus can accommodate andremove harmful sequences from the virus thereby. expanding the virus'sutility.

The technology described herein supports construction of deleted monkeyadenoviruses. As mentioned above the adenovirus E1 region encodes forregulatory proteins. In the absence of E1 function adenoviruses arereplication defective (also referred to herein as“replication-deficient”). The Examples below demonstrate that fullcomplementation of the replication deficiency of E1-deleted monkeyadenovirus is achieved with human adenovirus E1 and E4ORF6 (see Example3). Therefore, these regions essential for growth of monkey adenoviruson monkey cells are non-essential for growth in the human cellline—human adenovirus system (Example 3). The proteins encoded by the E3region are dispensable for virus growth. It is known in the art thatadenoviruses with E3 deletions are readily produced even in viruses withE1 and or E4 deletions. Surprisingly, of the multiple known E4 proteinsthe one responsible for expanded host range of monkey adenoviruses tohuman cells (ORF6) is also capable of complementing growth E4 deletedadenoviruses. Expression of both E1 and E4 from the same group Cserotype has been shown to support growth of non-species C adenoviruseswith E1-, E3-, E4-deletions (Tatsis et al., Gene Ther., 13(5): 421-9(2006)).

Adenovirus vectors derived from monkey adenoviruses are useful for thefollowing applications: (1) vaccine vectors for infectious diseaseindications, (2) vaccine vectors for anti-cancer applications, (3)transfer of transgenes encoding therapeutic proteins for acute andchronic disease intervention.

Monkey adenovirus vectors can be used for inducing immune responses(vaccination) in mammals. In this respect, widespread use of humanadenoviral vectors is hindered, at least in part, by the immunogenicityof the vector. A majority of the U.S. population has been exposed towild-type human adenovirus and developed pre-existing immunity to humanadenovirus-based gene transfer vectors. As a result, human adenoviralvectors are inactivated by the pre-existing host immune response,thereby reducing the effectiveness of the vector. The neutralizationand/or clearance of adenoviral vectors in the body complicates use ofthese vectors as DNA vaccines. DNA vaccines employ gene transfer vectorsto deliver antigen-encoding DNA to host cells. By producing antigenicproteins in vivo, the humoral and cell-mediated arms of the immunesystem are activated, thereby generating a more complete immune responseagainst the antigen as compared to traditional vaccines wherein foreignproteins are injected into the body. Despite the advantageouscharacteristics of human adenoviral vectors as gene delivery vehicles,the immunogenicity of the vector prevents efficient repeat dosing, whichcan be advantageous for “boosting” the immune system against pathogens,and results in only a small fraction of a dose of adenoviral vectordelivering its payload to host cells.

The monkey adenoviral vectors will not be subject to neutralizationand/or clearance mediated by pre-existing immunity to human adenovirus.Also, the combination of two or more monkey adenoviral vectors willcircumvent the inhibition seen with repeated administration of humanadenovirus vectors; thus it will he possible to boost the immune systemagainst pathogens. Thus, the monkey adenovirus vectors provide the sameadvantages of the human adenoviral vectors without their shortcomings.

One embodiment of the adenovirus of the invention comprises a nucleicacid sequence encoding an antigen which is expressed in the mammal toinduce an immune response. An “antigen” is a molecule that triggers animmune response in a mammal. An “immune response” can entail, forexample, antibody production and/or the activation of immune effectorcells. An antigen in the context of the invention can comprise anysubunit of any proteinaceous molecule, including a protein or peptide ofviral, bacterial, parasitic, fungal, protozoan, prion, cellular, orextracellular origin, which ideally provokes an immune response in amammal, preferably leading to protective immunity. The antigen also canbe a self-antigen, i.e., an autologous protein which the body hasmistakenly identified as a foreign invader.

In one embodiment, the antigen is a viral antigen. The viral antigen canbe isolated from any virus including, but not limited to, a virus fromany of the following viral families: Arenaviridae, Arterivirus,Astroviridae, Baculoviridae, Badnavirus, Barnaviridae, Birnaviridae,Bromaviridae, Bunyaviridae, Caliciviridae, Capillovirus, Carlavirus,Caulimovirus, Circoviridae, Closterovirus, Cornoviridae, Coronaviridae(e.g., Coronavirus, such as severe acute respiratory syndrome (SARS)virus), Corticoviridae, Cystoviridae, Deltavirus, Dianthovirus,Enamovirus, Filoviridae (e.g., Marburg virus and Ebola virus (e.g.,Zaire, Reston, Ivory Coast, or Sudan strain)), Flaviviridae, (e.g.,Hepatitis C virus, Dengue virus 1, Dengue virus 2, Dengue virus 3, andDengue virus 4), Hepadnaviridae (e.g., Hepatitis B virus), Herpesviridae(e.g., Human herpesvirus 1, 2, 3, 4, 5, and 6, and Cytomegalovirus),Hypoviridae, Iridoviridae, Leviviridae, Lipothrixviridae, Microviridae,Orthomyxoviridae (e.g., Influenzavirus A and B), Papovaviridae,Paramyxoviridae (e.g., measles, mumps, and human respiratory syncytialvirus), Parvoviridae, Picornaviridae (e.g., poliovirus, rhinovirus,hepatovirus, and aphthovirus), Poxviridae (e.g., vaccinia virus),Reoviridae (e.g., rotavirus), Retroviridae (e.g., lentivirus, such ashuman immunodeficiency virus (HIV) 1 and HIV 2), Rhabdoviridae, andTotiviridae. In one embodiment, an antigen of the inventive method is aRespiratory Syncytial Virus (RSV) antigen. The antigen can be, forexample, an RSV strain A or strain B antigen, such as all or part of theF, G, M, M1, M2, SH, or NS1, or NS2 proteins, or a fusion of all or partof more than one of these proteins. An antigen encoded by the adenoviralvector also can be a retroviral antigen. The retroviral antigen can be,for example, an HIV antigen, such as all or part of the gag, env, or polproteins. Any glade of HIV is appropriate for antigen selection,including clades A, B, C, MN, and the like. In some embodiments, atleast one antigen encoded by the adenoviral vector is a Herpes SimplexVirus 2 (HSV-2) antigen. Suitable SARS virus antigens for the inventivemethod include, for example, all or part of the UL19, UL47, or gDproteins. The antigenic peptides specifically recited herein are merelyexemplary as any viral protein can be used in the context of theinvention.

The antigen also can be a parasite antigen such as, but not limited to,a Sporozoan antigen. For example, the nucleic acid sequence can encode aPlasmodium antigen, such as all or part of a Circumsporozoite protein, aSporozoite surface protein, a liver stage antigen, an apical membraneassociated protein, or a Merozoite surface protein.

Alternatively or in addition, at least one antigen encoded by theadenoviral vector is a bacterial antigen. The antigen can originate fromany bacterium including, but not limited to, Actinomyces, Anabaena,Bacillus, Bacteroides, Bdellovibrio, Caulobacter, Chlamydia, Chlorobium,Chromatium, Clostridium, Cytophaga, Deinococcus, Escherichia,Halobacterium, Heliobacter, Hyphomicrobium, Methanobacterium,Micrococcus, Myobacterium, Mycoplasma, Myxococcus, Neisseria,Nitrobacter, Oscillatoria, Prochloron, Proteus, Pseudomonas,Phodospirillum, Rickettsia, Salmonella, Shigella, Spirillum,Spirochaeta, Staphylococcus, Streptococcus, Streptomyces, Sulfolobus,Thermoplasma, Thiabacillus, and Treponema. In one embodiment, at leastone antigen encoded by the nucleic acid sequence is a Pseudomonasantigen or a Heliobacter antigen.

It will be appreciated that an entire, intact viral or bacterial proteinis not required to produce an immune response. Indeed, most antigenicepitopes are relatively small in size and, therefore, protein fragmentscan be sufficient for exposure to the immune system of the mammal, Inaddition, a fusion protein can be generated between two or moreantigenic epitopes of one or more antigens. For example, all or part ofHIV gp120 or gp160 can be fused to all or part of the HIV pol protein togenerate a more complete immune response against the HIV pathogencompared to that generated by a single epitope. Delivery of fusionproteins by an adenoviral vector to a mammal allows exposure of animmune system to multiple antigens and, accordingly, enables a singlevaccine composition to provide immunity against multiple pathogens.

A nucleic acid sequence, including one encoding an antigen, is notlimited to a type of nucleic acid sequence or any particular origin. Thenucleic acid sequence can be recombinant DNA, can be genomic DNA, can beobtained from a DNA library of potential antigenic epitopes, or can besynthetically generated. The nucleic acid sequence can be present aspart of an expression cassette, which additionally comprises the geneticelements required for efficient expression of the nucleic acid sequenceand production of the encoded antigen. Ideally, an antigen-encodingnucleic acid sequence is operably linked to a promoter and apolyadenylation sequence as described herein. A promoter can be selectedfor use in a method of the invention by matching its particular patternof activity with the desired pattern and level of expression of theantigen(s). For example, an adenoviral vector can comprise two or morenucleic acid sequences that encode different antigens and are operablylinked to different promoters displaying distinct expression profiles.For example, a first promoter is selected to mediate an initial peak ofantigen production, thereby priming the immune system against an encodedantigen. A second promoter is selected to drive production of the sameor different antigen such that expression peaks several days after thatof the first promoter, thereby “boosting” the immune system against theantigen. Alternatively, a hybrid promoter can be constructed whichcombines the desirable aspects of multiple promoters. For example, aCMV-RSV hybrid promoter combining the CMV promoter's initial rush ofactivity with the RSV promoter's high maintenance level of activity isespecially preferred for use in many embodiments of the inventivemethod. In that antigens can be toxic to eukaryotic cells, it may beadvantageous to modify the promoter to decrease activity incomplementing cell lines used to propagate the adenoviral vector.

The following examples further illustrate the invention but, of course,should not be construed as in any way limiting its scope.

EXAMPLE 1

This example demonstrates that monkey adenovirus plaque formation ishighly efficient on a human cell line expressing human adenoviruscomponents E1 and E4ORF6.

Monkey adenoviruses are restricted from replication on human cells andhuman adenovirus factors overcome the restriction. As demonstrated herethe invention has two unexpected results: improved growth of monkeyadenoviruses on human cells over those that contain the entire E4region, and superiority in propagating monkey adenoviruses on humancells compared to their native host cell lines recommended for theirgrowth. Two methodologies were used to define growth. Plague formationwas used to determine growth under conditions of very low multiplicityof infection (MOI) and more than one infectious cycle is required togenerate a positive result, i.e., a plague in the cell monolayer. Thus,plague formation truly demonstrates continuous growth of a virus: singleinfectious viral particles infect a single cell, full completion of theviral life cycle occurs, followed by infection of neighboring cells bythe viral progeny and so on. The second method assesses the number ofinfectious viral progeny produced from a single round of viralreplication. This method is based on synchronous infection ofessentially all the cells in the monolayer, known as single-burst growthassessments,

Of all the E4 factors, it is demonstrated herein that ORF6 is sufficientto propagate monkey adenoviruses on human cells. Unexpectedly, the humancell line is superior to monkey cell lines in supporting the ability ofmonkey adenoviruses to form plaques, a standard method to measure virusgrowth. Two monkey adenoviruses, SV-11 and SV-38, which were isolatedfrom Rhesus and Vervet monkeys, respectively, were plagued 293-ORF6,BSC-1, LLC-MK2 (MK2), Vero, and CV-1 cells. All but the 293-ORF6 cellline are derived from monkeys. BSC-1 and MK-2 are the cell linesrecommended by the American Type Culture Collection (ATCC) to propagatethe viruses on. A serial dilution of both viruses was used to infecteach cell line, which were 80% confluent in 60 mm culture dishes. Theinfection conditions were 500 ul of virus for 1 hour rocked every 15minutes. The virus was subsequently removed and cells overlaid withEMEM+2% FBS and 0.9% agarose. Fourteen days later plaques were counted.293-ORF6 cells gave the highest plaguing efficiency being at least tentimes better than any of the other cells lines tested (Table 1).Previously it was reported that monkey virus plagued on human cells thatcontain the entire E4 region from human adenovirus 2 had a plagueformation efficiency 40 times lower compared to CV-1 cells. With thisinvention there is a clear 400-fold improvement in growth as measured byplague forming activity. As demonstrated here, the invention has twounexpected results: improved growth of monkey adenoviruses on humancells over those that contain the entire E4 region, and superiority inpropagating monkey adenoviruses on human cells compared to their nativehost cell lines recommended for their growth.

TABLE 1 Cell Line Virus Titer (PFU*/mL) BS-C-1 SV-11 1.67 × 10⁸ CV-1SV-11 2.89 × 10⁷ LLC-MK2 SV-11 2.54 × 10⁷ Vero SV-11 1.94 × 10⁸ 293-ORF6SV-11 6.36 × 10⁹ BSC-1 SV-38 1.27 × 10⁷ CV-1 SV-38 4.09 × 10⁶ LLC-MK2SV-38  1.0 × 10⁷ Vero SV-38 6.66 × 10⁶ 293-ORF6 SV-38 2.26 × 10⁸ *plaqueforming units

EXAMPLE 2

This example demonstrates the generation of high-titers of monkeyadenovirus viral progeny on a human cell line with human adenovirusspecies C factors.

Single-burst growth experiments were performed to determine if thecombination of human adenovirus E1 and E4 Orf6 was sufficient toovercome the restriction block to monkey adenovirus replication in humancells. Human cells expressing either no Ad5 factors (A549), Ad5 E4ORF6(A549-+Ad5 E4ORF6), Ad5 E1 (293), or Ad5 E1 and E4ORF6 (293-ORF6)wereplated in triplicate in 6-well plates at 1.5×10⁶ cells per well. BSC-1cells derived from African Green Monkey that are permissive for monkeyadenovirus replication were plated at 1×10⁵ cells per well. All cellswere kept in DMEM with 10% FCS and grown at 37° C., 5% CO₂. The nextday, cells were infected with CsCl₂ gradient-purified monkey adenovirusstocks at an MOI of three (FIG. 2A) or one (FIG. 2B) Focus Forming Unit(FFU)/cell in 300 pi per well for two hours, followed by aspiration andoverlay with 3 ml of DMEM with 5% FCS and 100 μM ZnCl₂ to permitinduction of E4ORF6 expression in A549+Ad5 E4ORF6 and 293-ORF6 cells.Cells were then incubated and harvested at 72 hours post-infection.Virus particles were released from cells by three freeze-thaw cyclesconsisting of alternating exposures to dry ice and 37° C. water bath.The number of progeny virions in the virus-cell lysates was assessedusing the FFU assay described in Vaccine, 25: 2074-2084 (2007).

The generation of infectious progeny with monkey adenoviruses on humancell lines was highest with 293-ORF6 cells, compared to A549 and A549cells expressing Ad5 E4 ORF6 (FIG. 2A). Similarly, generation of viralprogeny at an MOI of 1 was at least as efficient on 293-ORF6 cells as onthe monkey cell line BSC-1 (FIG. 2B). In some cases, between 10 to1,000-fold more viral progeny were produced from 293-ORF6 cells ascompared to BSC-1 cells. In contrast, monkey adenovirus replication on293 cells was less efficient than on BSC-1 cells. Therefore, thepresence of E1 and E4ORF6 overcame the host replication block in humancells. This was further substantiated with SV-1 which yielded 90,090particles/cell after purification when grown on 293-ORF6 cells. Finally,generation of infectious progeny on 293-ORF6 cells is up to 1,000-foldhigher than on BSC-1 cells, indicating that monkey adenovirus growth onhuman cells expressing E1 and E4ORF6 is significantly greater than onthe native host cell lines recommended for monkey adenovirus growth.

Taken together, plaque formation efficiency and single-burst growthanalyses of the monkey adenoviruses demonstrate that monkey adenovirusescan efficiently replicate in 293-ORF6 cells, and that the combinedexpression of human adenovirus components (Ads E1 and E4ORF6) overcomesthe human replication block. Furthermore, this was demonstrated witheight monkey adenoviruses, a significant representation of the group.

EXAMPLE 3

This example demonstrates the construction and propagation of monkeyadenovirus vectors with E1 deletions on a human cell line with humanadenovirus components.

Seven different monkey adenoviruses from three different species(Vervet, Cynomolgus, and Rhesus macaque) had their E1 region replacedwith an expression cassette. The monkey adenoviruses used here are SV-1,SV-7, SV-11, SV-16, SV-18, SV-38, and SV-39 from the ATCC. To facilitatetheir cloning the left ITR to E1a promoter, pIX promoter region andsequences next to the right ITR of the wild type viruses weredetermined. The E1 deletions were designed to inactivate the E1 apromoter and E1 proteins but retain the pIX viral promoter, viralpackaging signal and origin of replication. The identity of thesesequences was determined by their homology to known adenovirus genomes,and can be identified using publically available software, for example,as that found at the Berkeley Drosophila Genome Project, National Centerfor Biotechnology Information and Expert Protein Analysis System. Ingeneral, the E1 deletion initiated within 50 base pair (bp) 5′ of theE1a promoter TATA box and ended 50 to 300 bp 5′ of the pIX promoter.These are only examples of where the deletions can be made, since thedeletions can be changed depending on the application of the viralvector. For example the E1 deletion for the SV-38 derived vectorretained most of the E1a promoter demonstrating that complete removal ofthe E1a promoter is not required in order. to construct an adenovirusvector.

Although there are multiple ways to construct such viruses, itessentially involves two steps. The desired viral vector genome isgenerated in bacteria, followed by transfecting the genome into 293-ORF6cells to make viral particles.

The general procedure to make the viral vector genomes in bacteria isoutlined in FIG. 3, or minor variants of it. Using standard molecularbiology techniques known in the art, a shuttle plasmid is constructedwhich comprises the viral ITR (stippled boxes), packaging signal (Ψ), anexpression cassette that replaces the E1 region, and pIX sequences,which are followed by the viral right ITR. The expression cassette inthis example is comprised of the CMV promoter (arrow), open readingframe (orf) and SV40 polyadenylation signal (SV). The orientation andcomposition of the expression cassette is illustrative and not intendedto be limiting. The plasmid preferably contains a low copy numberbacterial origin of replication, such as p15, although other origins ofreplication can also be used. Inclusion of a gene that providesresistance to an antibiotic such as kanamycin (Kan r), for example, isuseful to select for bacteria that harbor the plasmid. To construct theadenovirus vector genome, the remainder of the viral genome from pIX toright ITR is cloned into the shuttle plasmid by homologousrecombination. To accomplish this, recombination competent bacteria suchas BJ5183 are transformed with both ˜50 ng of the shuttle plasmidrestricted with an endonuclease between the pIX and right ITR and 100 ngof purified viral genome. It is preferable to have at least 50 bp ofhomology each between the pIX and right ITR of the shuttle plasmid andviral genome for recombination (X). There are numerous methods known inthe art to identity the bacteria with the cloned adenovirus vectorgenome, including restriction digest, polymerase chain reaction (PCR),and DNA sequencing. Sequencing of the viral hexon gene for example canbe used to further confirm the identity of the adenovirus. Homology tothe following hexon sequences was used to further confirm the identitiesof the adenoviruses and their derivatives: SV-1 (SEQ ID NO:22); SV-7(SEQ ID NO:23); SV-11 (SEQ ID NO:24); SV-16 (SEQ ID NO:25); SV-18 (SEQID NO:26); SV-38 (SEQ ID NO:27); SV-39 (SEQ ID NO:28). Once the viralvector genome has been identified, the plasmid is used to transform aRec^(A−) bacterial strain such as DH10B from which the plasmid isamplified and purified.

Next, the second step, the conversion (rescue) of infectious virus fromthe genomic plasmids, was conducted as follows. Genomic plasmids weredigested with a restriction enzyme to release both ITRs front theplasmid backbone, purified by phenol:chloroform:isoamyl alcoholextraction and ethanol precipitation, and resuspended in 10 mM Tris, 1mM EDTA, pH 8.0. The 293-ORF6 cell line, ˜1.5˜10⁶ cells per 60 mm plate,were then transfected with 5 ug of the digested plasmid with Polyfectreagent (Qiagen). Five days post-transfection the cells were harvested,subjected to three freeze-thaw cycles, and the cell lysate was passagedonto fresh cells. Cell-virus lysate was serial passaged in this mannerat three to five day intervals until full cytopathic effect (c.p.e.) wasobserved. Purified adenovirus stocks were generated from infected cellsas described in Gall JG, et al. Rescue and production of vaccine andtherapeutic adenovirus vectors expressing inhibitory transgenes. MolBiotechnol. 35(3):263-73 (2007), PubMed PMID: 17652790. In briefinfected cells were collected and the culture medium discarded. Thecells were lysed in 25 mM Tris pH 7.5, 75 mM NaCl, 5 mM MgCl₂ buffer bythree freeze/thaw cycles and treated with Benzonase® at 100 units/mLovernight at room temperature. Cesium chloride isopycnic gradientcentrifugation was performed and total particle unit titer wasdetermined by absorbance at 260 nm as described in Mittereder, N., etal., Evaluation of the concentration and bioactivity of adenovirusvectors for gene therapy. J. Virol. 70:7498-7509 (1996). The activeparticle titers were determined in the fluorescent focus unit (FFU)assay with the Ad2 hexon antibody as described in Gall JG, et al. Rescueand production of vaccine and therapeutic adenovirus vectors expressinginhibitory transgenes. Mol Biotechnol. 35(3):263-73 (2007), PubMed PMID:17652790.; and Lemiale F, et al., Novel adenovirus vaccine vectors basedon the enteric-tropic serotype 41. Vaccine 25(10:2074-84 (2007), Epub2006 Nov 28. PubMed PMID: 17250935, PubMed Central PMCID: PMC2584667.The viral preparations are of high quality with an average ratio oftotal particles to infectious particles of 49+/−23 (n=6) and highyields, with up to 37,000 particles per cell post-purification, Theidentity of each virus was confirmed by partial DNA sequencing anddiagnostic PCR. The functionality of the expression cassette wasconfirmed by Western blot analysis.

The identity of the viral sequences used to make the rescue shuttleplasmids described herein and in FIG. 3 are shown below. All sequencesare given in left to right orientation of the standard viral genome. Inthe standard viral genome the E1 region is on the left hand end of theviral genome. The sequence comprising the left hand ITR, packagingsignal but devoid of the complete E1a promoter is called “Left handsequence.” The sequence used for homologous recombination in the pIXregion is called “pIX region sequence.” The sequence that is comprisedof part of the right ITR that is used for homologous recombination iscalled “Right sequence.” All sequences are given in the standard 5′ to3′ direction.

SV-1: Left hand sequence: (SEQ ID NO:1); pIX region sequence: (SEQ IDNO:2); Right sequence: (SEQ ID NO:3).

SV-7: Left hand sequence: (SEQ ID NO:4); pIX region sequence: (SEQ IDNO:5); Right sequence: (SEQ ID NO:6).

SV-11: Left hand sequence: (SEQ ID NO:7); pIX region sequence: (SEQ IDNO:8); Right sequence: (SEQ ID NO:9).

SV-16: Left hand sequence: (SEQ ID NO:10); pIX region sequence: (SEQ IDNO:11); Right sequence: (SEQ ID NO:12).

SV-18: Left hand sequence: (SEQ ID NO:13); pIX region sequence: (SEQ IDNO:14); Right sequence: (SEQ ID NO:15).

SV-38: Left hand sequence: (SEQ ID NO:16); pIX region sequence: (SEQ IDNO:17); Right sequence: (SEQ ID NO:18).

SV-39: Left hand sequence: (SEQ ID NO:19); pIX region sequence: (SEQ IDNO:20); Right sequence: (SEQ ID NO:21).

EXAMPLE 4

This example demonstrates that monkey adenoviruses with deletion of E1region sequences are replication-deficient.

E1-deletions were engineered into the monkey adenoviruses SAV7, SAV11,and SAV16, as described in Example 3. The E1-deletions were designed toremove the promoter of E1A, the entirety of the E1 A coding region, theE1B promoter, the E1B 21K protein homolog coding region, and themajority of the 5′ end of the E1B 55K homolog coding region.Additionally, an expression cassette, as described in Example 3 and GallJG, et al., Rescue and production of vaccine and therapeutic adenovirusvectors expressing inhibitory transgenes. Mol. Biotechnol. 35(3):263-73(2007), PubMed PMID: 17652790, without an encoded protein product wasinserted into the location of the E1 deletion. To determine if theremoval of these sequences changed the replication of the viruses,monkey cells were infected with the viruses and the infectious viralprogeny were measured. Two cell lines both of monkey origin were used,and were recommended for progagation of the monkey adenoviruses by thevendor (ATCC). The LLC-MK2 cell line is a kidney line from rhesusmacaque (Macaca mullata), and the BSC-1 cell line is a kidney line fromAfrican green monkey (Cercopithecus aethiops). Adherent cultures of thecell lines, plated at 3×10⁵ cells per well of 6-well plates, wereinfected with 100 particles per cell of each of the E1-deleted and wildtype adenoviruses. At 72 and 96 hours post-infection (hpi), the cellsand medium were collected and subjected to three cycles of freezing andthawing (frozen on dry ice ˜10 minutes, thawed in a 37° C. water bath˜10 minutes) to lyse the cells. The cell lysates were assayed forinfectious virus in the fluorescent focus unit (FFU) assay as describedin Gall JG, et al. Rescue and production of vaccine and therapeuticadenovirus vectors expressing inhibitory transgenes. Mol. Biotechnol.35(3):263-73 (2007), PubMed PMID: 17652790.; and Lemiale F, et al.,Novel adenovirus vaccine vectors based on the enteric-tropic serotype41. Vaccine 25(11):2074-84 (2007), Epub 2006 Nov 28. PubMed PMID:17250935, PubMed Central PMCID: PMC2584667. There were high titers ofviral progeny in the lysates generated from cells infected with the wildtype viruses (Table 2); thus the cell cultures were permissive to thesimian adenoviruses. Importantly, there were no progeny virions detectedin the lysates from the cells infected with the E1-deleted simianadenoviruses, with an assay quantitation limit of 25,325 25,875 FFU mL(minimum of 5 foci per microscopic field, 1013 fields per cell culturewell, and undiluted lysate). Comparisons of the maximum titers achievedwith each serotype of wild type virus to the quantitation limit showsreduced replication of SAV7 by at least a factor of 19,348; of SAV11 byat least a factor of 10,266; and of SAV16 by at least a factor of 5,133.In addition, there were no foci observed in wells infected with thelysates from the null virus infections, further lowering the assay limitto the limit of detection, 5 FFU/mL. Comparison of the maximum titersachieved with each serotype of wild type virus to the detection limitestablishes reduced replication by a factor of 9.8×10⁷, 5.2×10⁷, and2.6×10⁷ for SAV7, SAV11, and SAV16, respectively. Therefore, the simianadenoviruses with the E1-deletions were replication-deficient.

TABLE 2 Generation of viral progeny (FFU/mL). BSC-1 LLC-MK2 Virus 72 hpi96 hpi 72 hpi 96 hpi SAV7 null 0 0 0 0 SAV11 null 0 0 0 0 SAV16 null 0 00 0 SAV7 WT 2.5E+08 3.7E+08 2.7E+07 4.9E+08 SAV11 WT 2.6E+08 n.d.*1.9E+08 n.d. SAV16 WT 5.7E+06 1.3E+08 2.0E+06 5.0E+06 *n.d. = not done.

All references, including publications, patent applications, andpatents, cited herein are hereby incorporated by reference to the sameextent as if each reference were individually and specifically indicatedto be incorporated by reference and were set forth in its entiretyherein.

The use of the terms “a” and “an” and “the” and similar referents in thecontext of describing the invention (especially in the context of thefollowing claims) are to be construed to cover both the singular and theplural, unless otherwise indicated herein or clearly contradicted bycontext. The terms “comprising,” “having,” “including,” and “containing”are to be construed as open-ended terms (i.e., meaning “including, butnot limited to,”) unless otherwise noted. Recitation of ranges of valuesherein are merely intended to serve as a shorthand method of referringindividually to each separate value falling within the range, unlessotherwise indicated herein, and each separate value is incorporated intothe specification as if it were individually recited herein. All methodsdescribed herein can be performed in any suitable order unless otherwiseindicated herein or otherwise clearly contradicted by context. The useof any and all examples, or exemplary language (e.g., “such as”)provided herein, is intended merely to better illuminate the inventionand does not pose a limitation on the scope of the invention unlessotherwise claimed, No language in the specification should be construedas indicating any non-claimed element as essential to the practice ofthe invention.

Preferred embodiments of this invention are described herein, includingthe best mode known to the inventors for carrying out the invention.Variations of those preferred embodiments may become apparent to thoseof ordinary skill in the art upon reading the foregoing description. Theinventors expect skilled artisans to employ such variations asappropriate, and the inventors intend for the invention to be practicedotherwise than as specifically described herein, Accordingly, thisinvention includes all modifications and equivalents of the subjectmatter recited in the claims appended hereto as permitted by applicablelaw, Moreover, any combination of the above-described elements in allpossible variations thereof is encompassed by the invention unlessotherwise indicated herein or otherwise clearly contradicted by context.

We claim:
 1. A method of propagating a monkey adenovirus, which methodcomprises contacting a cell with the monkey adenovirus, wherein the cellexpresses a gene product encoded by one or both of the E1A region andthe E1B region, and a gene product encoded by a portion of the E4 regionconsisting essentially of E4 ORF6, of a human adenovirus, whereby themonkey adenovirus is propagated in the cell, wherein monkey refers toboth new world and old world monkeys, and does not include any member ofthe family Hominidae.
 2. The method of claim 1, wherein the humanadenovirus is a species C human adenovirus.
 3. The method of claim 2,wherein the portion of the E4 region is E4 ORF6.
 4. The method of claim3, wherein the cell is a human cell.
 5. The method of claim 4, whereinthe cell is a HEK-293 cell or a PerC.6 cell.
 6. The method of any one ofclaims 1-5, wherein the monkey adenovirus is replication-deficient. 7.The method of claim 6, wherein the monkey adenovirus requirescomplementation of one or more of the E1A region, the E1B region, andthe E4 region of the adenoviral genome for propagation.
 8. The method ofclaim 7, wherein the monkey adenovirus comprises a deficiency in the E1region and a deficiency in at least a portion of the E4 region of theadenoviral genome.
 9. The method of claim 8, wherein the monkeyadenovirus further comprises a deficiency in the E3 region of theadenoviral genome.